Phosphoproteomics reveals distinct modes of Mec1/ATR signaling during DNA replication.

The Mec1/Tel1 kinases (human ATR/ATM) play numerous roles in the DNA replication stress response. Despite the multi-functionality of these kinases, studies of their in vivo action have mostly relied on a few well-established substrates. Here we employed a combined genetic-phosphoproteomic approach to monitor Mec1/Tel1 signaling in a systematic, unbiased, and quantitative manner. Unexpectedly, we find that Mec1 is highly active during normal DNA replication, at levels comparable or higher than Mec1's activation state induced by replication stress. This "replication-correlated" mode of Mec1 action requires the 9-1-1 clamp and the Dna2 lagging-strand factor and is distinguishable from Mec1's action in activating the downstream kinase Rad53. We propose that Mec1/ATR performs key functions during ongoing DNA synthesis that are distinct from their canonical checkpoint role during replication stress.

Mitochondrial superoxide dismutase Sod2 suppresses nuclear genome instability during oxidative stress.

Oxidative stress can damage DNA and thereby contribute to genome instability. To avoid an imbalance or overaccumulation of reactive oxygen species (ROS), cells are equipped with antioxidant enzymes that scavenge excess ROS. Cells lacking the RecQ-family DNA helicase Sgs1, which contributes to homology-dependent DNA break repair and chromosome stability, are known to accumulate ROS, but the origin and consequences of this oxidative stress phenotype are not fully understood. Here, we show that the sgs1 mutant exhibits elevated mitochondrial superoxide, increased mitochondrial mass, and accumulation of recombinogenic DNA lesions that can be suppressed by antioxidants. Increased mitochondrial mass in the sgs1Δ mutant is accompanied by increased mitochondrial branching, which was also inducible in wildtype cells by replication stress. Superoxide dismutase Sod2 genetically interacts with Sgs1 in the suppression of nuclear chromosomal rearrangements under paraquat (PQ)-induced oxidative stress. PQ-induced chromosome rearrangements in the absence of Sod2 are promoted by Rad51 recombinase and the polymerase subunit Pol32. Finally, the dependence of chromosomal rearrangements on the Rev1/Pol ζ mutasome suggests that under oxidative stress successful DNA synthesis during DNA break repair depends on translesion DNA synthesis.

Maintenance of Yeast Genome Integrity by RecQ Family DNA Helicases.

With roles in DNA repair, recombination, replication and transcription, members of the RecQ DNA helicase family maintain genome integrity from bacteria to mammals. Mutations in human RecQ helicases BLM, WRN and RecQL4 cause incurable disorders characterized by genome instability, increased cancer predisposition and premature adult-onset aging. Yeast cells lacking the RecQ helicase Sgs1 share many of the cellular defects of human cells lacking BLM, including hypersensitivity to DNA damaging agents and replication stress, shortened lifespan, genome instability and mitotic hyper-recombination, making them invaluable model systems for elucidating eukaryotic RecQ helicase function. Yeast and human RecQ helicases have common DNA substrates and domain structures and share similar physical interaction partners. Here, we review the major cellular functions of the yeast RecQ helicases Sgs1 of Saccharomyces cerevisiae and Rqh1 of Schizosaccharomyces pombe and provide an outlook on some of the outstanding questions in the field.